Selective Activation of PPARα Mitigates Peritoneal Inflammation and Fibrosis through NLRP3 Inflammasome Suppression and Inflammation Modulation

doi:10.21203/rs.3.rs-4003336/v1

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Selective Activation of PPARα Mitigates Peritoneal Inflammation and Fibrosis through NLRP3 Inflammasome Suppression and Inflammation Modulation

https://doi.org/10.21203/rs.3.rs-4003336/v1

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published 11 Oct, 2024

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Peritoneal inflammation and fibrosis remain major challenges to the long-term maintenance of peritoneal dialysis. Pemafibrate, a selective peroxisome proliferator-activated receptor α (PPARα) modulator, has been implicated in the management of fibrosis-related disorders. We investigated whether pemafibrate ameliorates peritoneal inflammation and fibrosis and explored the underlying mechanisms in mice with methylglyoxal (MGO)-induced peritoneal fibrosis (MGO mice). MGO mice exhibited peritoneal fibrosis with increased expression of mesenchymal markers, transforming growth factor-β1 (TGF-β1), and substantial deposition of extracellular matrix (ECM) proteins. Additionally, MGO mice exhibited peritoneal inflammation as indicated by elevated tumor necrosis factor-α expression and macrophage infiltration in peritoneal tissue. These effects were mitigated by pemafibrate treatment, which also restored peritoneal membrane function. Furthermore, pemafibrate promoted anti-inflammatory macrophage polarization in both mice and THP-1 cells. In human peritoneal mesothelial cells (HPMCs), pemafibrate effectively inhibited interferon-γ-induced production of TGF-β1 and ECM while suppressing the proinflammatory cytokines nuclear factor-κB (NF-κB) and activator protein 1. The NF-κB inhibitory effect of pemafibrate involved stabilization of the NF-κB inhibitory protein IkBα. Notably, pemafibrate hindered activation of the NLR family pyrin domain containing 3/caspase-1 axis in interferon-γ-stimulated HPMCs. These findings suggest that pemafibrate ameliorates peritoneal inflammation and fibrosis, making it a promising candidate for peritoneal fibrosis therapy.

Health sciences/Nephrology/Renal replacement therapy/Peritoneal dialysis

Biological sciences/Immunology/Inflammation

peritoneal dialysis

peritoneal fibrosis

peritoneal inflammation

PPARα

inflammasome

Peritoneal dialysis (PD) is a well-established home-based treatment for kidney failure and is performed in approximately 9% of patients undergoing renal replacement therapy worldwide ¹. However, long-term PD has been shown to cause peritoneal fibrosis, which is accompanied by morphological and functional changes in the peritoneum due to inflammation induced by bio-incompatible PD fluid and infection ^2,3. In particular, peritoneal fibrosis results in not only structural alterations but also reduced efficacy of PD function and the onset of ultrafiltration failure, ultimately leading to discontinuation of PD ^4,5. Peritoneal fibrosis is also considered a risk factor for the development of encapsulating peritoneal sclerosis, the most severe complication of PD, with potentially fatal consequences ⁶. Pathophysiologically, peritoneal fibrosis is associated with loss of mesothelial cells, increased collagen accumulation, myofibroblast proliferation, infiltration of inflammatory cells into the submesothelial region, and angiogenesis ². Prolonged exposure to glucose, glucose degradation products, and advanced glycation end products leads to the production of various cytokines. These include transforming growth factor-β (TGF-β) and interferon-γ (IFN-γ), which play a central role in the pathogenesis of peritoneal fibrosis ^7–10. However, the underlying mechanisms of peritoneal fibrosis remain incompletely understood, and effective treatments have yet to be established.

Peroxisome proliferator-activated receptor α (PPARα) belongs to the steroid/nuclear receptor superfamily and is predominantly expressed in highly active metabolic tissues, including the kidneys, liver, and heart ^11,12. Ligand-induced activation of PPARα signaling combines with retinoid X receptor to form a heterodimer, activate the PPAR response element located upstream of the target gene, and regulate the expression of genes involved in lipid metabolism and inflammation ^13,14. Indisputable evidence of the role of PPARα in anti-inflammatory activities was obtained when PPARα-deficient mice exhibited a delayed response to inflammatory stimuli ¹⁵. Although its mechanism has not been fully elucidated, PPARα has been shown to interfere with the transcriptional activity of inflammatory factors, including nuclear factor-κB (NF-κB) and activator protein 1 (AP-1) ^16,17. Pemafibrate is a novel selective PPARα modulator with higher potency and selectivity than conventional fibrates ^18–20. Numerous reports have established the involvement of PPARα in the pathogenesis of fibrosis in various organs ^21–24. Therefore, in the present study, we evaluated the role of pemafibrate in peritoneal inflammation and fibrosis.

Inflammasomes are a group of multimeric protein complexes that consist of nucleotide-binding oligomerization domain-like receptor (NLR) proteins and adaptors. The function of these NLR proteins and adaptors is to detect infectious or noxious stimuli and activate specific inflammatory caspase proteases, most notably caspase-1, leading to maturation of the inflammatory cytokines interleukin (IL)-1b and IL-18 ^25,26. The NLR family pyrin domain containing 3 (NLRP3) inflammasome is currently the most fully characterized inflammasome ²⁷. NLRP3 triggers the pathogenesis of complex diseases such as chronic kidney disease ^28,29 and type 2 diabetes ³⁰. The NLRP3 inflammasome was recently implicated in the pathogenesis of peritoneal injury and fibrosis ^31,32. Interestingly, pemafibrate reduces inflammasome activation and recovers fibrosis in the liver and heart ^33,34. However, the role of pemafibrate in regulating the NLRP3 inflammasome activity in PD-associated peritoneal fibrosis remains to be elucidated.

In the present report, we describe the involvement of PPARα in the pathogenesis of methylglyoxal (MGO)-induced peritoneal fibrosis. We then demonstrate that pemafibrate ameliorates peritoneal fibrosis and enhances peritoneal function, concurrently mitigating peritoneal tissue inflammation mediated by NF-κB and AP-1. Additionally, we illustrate that this reduction in inflammation corresponds with alterations in macrophage polarization. Finally, our findings substantiate that pemafibrate-induced attenuation of peritoneal fibrosis involves regulation of the NLRP3 inflammasome. These coherent outcomes corroborate the proposition that the suppressive effect of pemafibrate on peritoneal fibrosis involves NLRP3 inflammasome suppression, providing novel insights into the pathogenesis of this disorder and presenting new therapeutic avenues.

Enhancement of PPARα expression by pemafibrate suppressed MGO-induced peritoneal cell accumulation and thickening in mice

Several studies have shown that impaired PPARα function plays a significant role in tissue fibrosis ^35,36. We therefore hypothesized that pemafibrate, a potent and selective PPARα modulator ¹⁹, might also have a suppressive effect on peritoneal fibrosis. As depicted in Fig. 1A, peritoneal fibrosis was induced using the well-established MGO-injected mouse model ^37,38. To assess the impact of pemafibrate on PPARα expression in MGO-induced peritoneal fibrosis, we first performed immunohistochemical staining of PPARα. As shown in Fig. 1B and E, control mice exhibited few PPARα-positive cells in the mesothelial zone. By contrast, MGO-injected mice showed higher numbers of PPARα-positive cells in the mesothelial zone. Moreover, pemafibrate-treated mice displayed even higher numbers of PPARα-positive cells in the mesothelial zone than the MGO-injected mice.

We conducted hematoxylin–eosin staining and Masson’s trichrome staining to investigate the morphological changes caused by pemafibrate administration. In the MGO-injected mice, the peritoneal cell density was increased and submesothelial compact zone was thickened. However, pemafibrate administration in the MGO-injected mice significantly suppressed both the number of cells (Fig. 1C, F) and the thickening of the submesothelial compact zone (Fig. 1D, G) compared with the MGO-injected mice.

Pemafibrate inhibited MGO-induced fibrosis in mice with peritoneal fibrosis

To better understand the effect of pemafibrate in peritoneal fibrosis, we examined the expression of profibrotic markers in peritoneal tissue. Immunohistochemical staining showed that MGO injection remarkably elevated the number of α-SMA-positive myofibroblasts and FSP-1-positive fibroblasts in the submesothelial compact zone. By contrast, pemafibrate administration significantly reduced MGO-induced α-SMA-positive myofibroblasts (Fig. 2A, F) and FSP-1-positive fibroblasts (Fig. 2B, G) compared with the MGO-injected mice. The expression of collagen I and III was increased in the submesothelial compact zone of MGO-injected mice (Fig. 2C, H). However, pemafibrate administration significantly diminished collagen I and III accumulation compared with the MGO-injected mice (Fig. 2D, I). Moreover, the number of TGF-β1-positive cells in the MGO-injected mice was markedly higher than that in the control mice, whereas pemafibrate administration significantly reduced the number of increased TGF-β1-positive cells (Fig. 2E, J). Similarly, ELISA demonstrated that pemafibrate administration lowered the elevated TGF-β1 protein levels in mouse peritoneal fluid (Fig. 2K).

Pemafibrate inhibited inflammatory cytokine production and macrophage accumulation in mice

In several mouse models, pemafibrate has demonstrated preventive effects against inflammation ^33,39. To assess the effects of pemafibrate administration on peritoneal inflammation, we therefore examined the expression of inflammatory marker TNF-α and pan-macrophage marker CD68 in peritoneal tissues. Immunohistochemical staining demonstrated that TNF-α expression was increased in the submesothelial compact zone of MGO-injected mice. However, pemafibrate administration significantly diminished MGO-induced TNF-α expression (Fig. 3A, C). Additionally, MGO injection remarkably increased the number of CD68-positive macrophages in the submesothelial compact zone. By contrast, pemafibrate administration significantly reduced the increased number of CD68-positive macrophages (Fig. 3B, D). Furthermore, ELISA demonstrated that pemafibrate administration significantly inhibited the induction of IL-1β protein levels in mouse peritoneal fluid (Fig. 3E).

Pemafibrate enhanced macrophage to an anti-inflammatory macrophage phenotype and inhibited inflammatory markers in THP-1 cells

Macrophages play a central role in inflammatory cytokine production in the abdominal cavity ⁴⁰. These cells exist on a continuum of activation states, with classically activated (M1, proinflammatory) macrophage characterized by secretion of proinflammatory cytokines and alternatively activated (M2, anti-inflammatory) macrophage characterized by secretion of anti-inflammatory cytokines ⁴¹. To investigate the role of pemafibrate in macrophage polarization, we examined the expression of CD163, an established anti-inflammatory macrophage marker, in mice. Immunohistochemical staining showed that control mice exhibited almost no CD163-positive cells in the submesothelial compact zone (Fig. 4A, B). However, MGO-injected mice exhibited increased CD163-positive cells, and pemafibrate administration resulted in an even higher number of CD163-positive cells compared with the MGO-injected mice (Fig. 4A, B).

We evaluated the expression of CD163 in THP-1 cells to provide direct evidence of the role of pemafibrate in anti-inflammatory macrophage polarization. IFN-γ, a cytokine primarily produced by immune cells and involved in immune and inflammatory responses, is recognized for its role in regulating proinflammatory macrophage polarization ⁴². Furthermore, MGO injection demonstrated a significant increase in IFN-γ expression within the mice peritoneum (Figure S1A, B). Therefore, we proceeded to stimulate cells with IFN-γ. Western blotting showed that IFN-γ alone was not sufficient to induce anti-inflammatory polarization in macrophages (Fig. 4C–E). Interestingly, pemafibrate treatment promoted anti-inflammatory polarization. We next examined the levels of inflammatory cytokines in the cultured supernatant to confirm the effect of pemafibrate on anti-inflammatory macrophage polarization in THP-1 cells. ELISA showed that the protein levels of TNF-α and IL-1β in the pemafibrate pretreatment group were significantly decreased (Fig. 4F, G).

Pemafibrate reduced peritoneal membrane functional impairments in mice with peritoneal fibrosis

On day 21, we performed a PET to elucidate the functional alteration of the peritoneal membrane in mice. The D/P ratio of BUN and the peritoneal absorption of glucose from the dialysate obtained during the PET (D/D0) were assessed. The transport rate of BUN from plasma and the absorption rate of glucose from dialysate were markedly higher in MGO-injected mice than in control mice, whereas these changes were significantly alleviated in pemafibrate-treated mice (Fig. 5A, B).

Pemafibrate inhibited IFN-γ-induced fibrotic and inflammatory markers in HPMCs

The pivotal involvement of peritoneal mesothelial cells in peritoneal fibrosis and subsequent functional decline of the peritoneal membrane is well-established ⁹. We investigated the expression of fibrotic markers in HPMCs to further clarify the direct impact of pemafibrate on peritoneal fibrosis. Western blotting revealed that stimulation with IFN-γ led to elevated protein levels of TGF-β1 and increased secretion of fibronectin in HPMCs. Conversely, treatment with pemafibrate effectively attenuated the increased levels of TGF-β1 protein as well as the elevated expression of secreted fibronectin (Fig. 6A, B).

Pemafibrate downregulates the transcription factor NF-κB activity, which influences inflammatory cytokines, and one hypothesis explaining this mechanism suggests stabilization of the NF-κB inhibitor protein IkBα ¹⁷. In our in vitro study, administration of pemafibrate to HPMCs induced by IFN-γ, promoting inflammatory responses, resulted in stabilization of IkBα and inhibition of NF-κB phosphorylation (Fig. 6C, D). Moreover, although its mechanism has not been fully elucidated, PPARα is known to interfere with the transcriptional activity of AP-1 in human aortic smooth muscle cells ¹⁶. In the present study, pemafibrate also inhibited the IFN-γ-induced expression of phospho-cJun (a component of AP-1).

An inflammasome is a mediator of caspase-1 activation that promotes the maturation of IL-1β ⁴³, and NF-κB is essential for accelerating the transcription of pro-IL-1β and NLRP3 inflammasomes ⁴⁴. Additionally, AP-1 promotes pro-IL-1β transcription to facilitate NLRP3 inflammasome activation ⁴⁵, synergically leading to tissue inflammation. As shown in Fig. 3E, administration of pemafibrate significantly inhibited the elevation of IL-1β protein levels in mouse peritoneal fluid. We thus assessed the levels of NLRP3 inflammasome and caspase-1 to further investigate the mechanism of the anti-inflammatory effect of pemafibrate. Western blotting demonstrated that IFN-γ induced NLRP3 inflammasome and caspase-1 expression and that pemafibrate inhibited IFN-γ-induced NLRP3 inflammasome and caspase-1 expression in HPMCs (Fig. 6F, G), thus collectively attenuating the inflammatory response.

This study revealed five significant findings. First, pemafibrate effectively enhances PPARα expression within the peritoneal tissue of mice while concurrently suppressing peritoneal fibrosis. Second, pemafibrate exhibits inhibitory effects on the expression of TNF-α, leading to a reduction in the number of CD68-positive cells within the peritoneal tissue of mice. Third, pemafibrate demonstrates a propensity to induce anti-inflammatory macrophage polarization both in mouse peritoneal tissue and THP-1 cells. Fourth, pemafibrate treatment substantially attenuates the elevated transport rate of BUN from plasma and the absorption rate of glucose from dialysate in mice, indicating its therapeutic potential. Finally, pemafibrate not only inhibits the expression of NF-κB and AP-1 but also suppresses activation of the NLRP3 inflammasome and subsequent caspase-1 inflammatory responses in HPMCs, explaining its anti-inflammation mechanism. These findings collectively suggest that the activation of PPARα holds promise as a potential therapeutic target for patients with peritoneal fibrosis. Furthermore, our study provides valuable insight into the underlying mechanism by which pemafibrate mitigates peritoneal fibrosis in a mouse model of MGO-induced peritoneal fibrosis.

Pemafibrate inhibited peritoneal dysfunction and fibrosis in this mouse model of MGO-induced chronic peritoneal disease. PPARα signaling pathways play a pivotal role in fibrotic-related diseases, which are associated with pathological proliferation of α-SMA-positive myofibroblasts and collagen deposition within the ECM ^2,46. These pathologic changes lead to functional and structural abnormalities in various organs and tissues, including the heart ⁴⁷, liver ²⁴, kidney ^23,35, and lung ⁴⁸. Pemafibrate was recently shown to be a high-affinity selective PPARα modulator with a superior safety profile ²⁰. To investigate the role of PPARα in the pathophysiology of peritoneal fibrosis in the present study, we prepared a well-established in vivo mouse model of MGO-induced peritoneal fibrosis as described above ³⁷. As expected, the activation of PPARα transcriptional activity by pemafibrate effectively mitigated peritoneal structural damage, ECM deposition, and peritoneal functional impairment induced by MGO injection, emphasizing the role of PPARα in the treatment of peritoneal fibrosis. Furthermore, the direct anti-fibrotic effect of pemafibrate was confirmed in IFN-γ-stimulated HPMCs. Taken together, these findings indicate that pemafibrate plays an important role in the pathogenesis of peritoneal fibrosis.

We have also demonstrated that pemafibrate ameliorates peritoneal inflammation, including a reduction in the expression of TNF-α and the number of CD68-positive macrophages in the submesothelial compact zone, as well as a decrease in the expression of IL-1β in mouse PD effluent. Our data are in line with previous studies showing that PPARα agonists exert anti-inflammatory effects as demonstrated using a PPARα-deficient mouse model ¹⁵. PPARα inhibits NF-κB, a master regulator of inflammation ^17,49, thereby suppressing the expression of TNF-α, IL-1β, and other cytokines ⁵⁰. Additionally, PPARα agonists alleviate peritoneal inflammation by modulating neutrophil and macrophage infiltration ⁵¹. The current data, coupled with the evidence of PPARα agonists exerting anti-inflammatory effects, therefore support the potential therapeutic benefits of pemafibrate in treating peritoneal fibrosis.

The NLRP3 inflammasome is currently the most fully characterized inflammasome ²⁷. Assembly of the NLRP3 inflammasome leads to caspase 1-dependent release of the proinflammatory cytokine IL-1β ²⁶. Interestingly, PPARα regulates inflammasome activity not only by interfering with NF-κB ^44,49 but also by affecting AP-1 ^16,52,53 transactivation capacity, contributing to its anti-inflammatory effects ^16,54. Specifically, activated PPARα interacts with both cJun, a major component of AP-1, and the p65 subunit of NF-κB through its binding to PPAR response elements as a heterodimer with the retinoid X receptor, thereby inhibiting AP-1- and NF-κB-mediated signaling ^16,55. Additionally, PPARα induces the inhibitory protein IκBα, which normally retains NF-κB in a nonactive form, leading to suppression of NF-κB DNA-binding activity ¹⁷. In the current study, pemafibrate reduced the proinflammatory cytokine IL-1β in mouse ascitic fluid and reduced activation of the NLRP3 inflammasome in HPMCs, which is consistent with these previous findings. Moreover, our data are consistent with a previous study showing that fenofibrate, a PPARα agonist, inhibited IL-1β maturation and caspase-1 activation in a monosodium urate-induced peritonitis mouse model ⁵⁶. Taken together, our findings demonstrate that pemafibrate contributes to inhibition of the NLRP3 inflammasome activation by suppressing NF-kB and AP-1, extending the functional scope of pemafibrate in the regulation of tissue inflammation.

Macrophage polarization is a frequently employed concept in the pathogenesis of inflammation. Differentiation programs implicated in generating distinct macrophage phenotypes have traditionally been categorized into two broad classes: classically activated (M1, proinflammatory) macrophages, which produce proinflammatory cytokines, and alternatively activated (M2, anti-inflammatory) macrophages, which are associated with anti-inflammatory properties ^41,57. We have herein demonstrated that pemafibrate treatment is implicated in the suppression of inflammation by facilitating polarization from the proinflammatory to anti-inflammatory macrophage phenotype in MGO-induced peritoneal inflammation. An anti-inflammatory macrophage polarization in this study was evidenced by an increase in the number of CD163-positive anti-inflammatory macrophages in immunohistochemistry analyses and an increase in the ratio of CD163-positive anti-inflammatory macrophages to CD68-positive pan-macrophages in Western blotting analyses, in accordance with our previous reports ^58,59. A previous study indicated that activation of PPARα in proinflammatory type-polarized macrophages inhibits the expression of proinflammatory cytokines, including TNF-α and IL-1β ⁶⁰. Additionally, Penas et al. demonstrated that PPARα plays a role in the polarization of macrophages isolated from Trypanosoma cruzi-infected mice, as evidenced by reduced expression of nitric oxide synthase 2 and proinflammatory cytokines and an increase in anti-inflammatory markers ⁶¹. Nonetheless, it is crucial to acknowledge that the classification of macrophages into proinflammatory (M1) and anti-inflammatory (M2) phenotypes in vivo may constitute a substantial oversimplification of the intricate spectrum of macrophage polarization states ⁶². Further studies are needed to prove these findings in the context of macrophage phenotypes in vivo and their impact on the microenvironment, particularly concerning the effects of pemafibrate treatment. Collectively, pemafibrate suppresses peritoneal inflammation, at least in part, by inducing the polarization of macrophages toward an anti-inflammatory phenotype.

In summary, our data provide clear, compelling evidence for the potential of pemafibrate, a PPARα-selective modulator, as a therapeutic agent for peritoneal fibrosis through the suppression of peritoneal inflammation. It highlights its multifaceted effects, including its anti-fibrotic effects and anti-inflammatory benefits. Moreover, our findings shed light on the intricate mechanisms by which pemafibrate exerts its protective effects in a mouse model of MGO-induced peritoneal fibrosis. Our data also suggest that PPARα-targeting agents ameliorate excessive NLRP3 activation via NF-kB and AP-1 activity, making them promising candidates for treatment of human fibrotic and inflammatory diseases.

Animals

Male C57BL/6J mice (age of 8 weeks, weight of 20–25 g) were purchased from Charles River Laboratories Japan (Yokohama, Japan). A total of 15 male mice were used in this study. The mice were housed in a light- and temperature-controlled room in the Laboratory Animal Center of Hiroshima University (Hiroshima, Japan) with free access to food and water. They were randomly divided into three groups (n = 5 in each group): control, MGO, and MGO + pemafibrate groups. All animal experiments were performed in accordance with the guidelines and with the approval of the Institutional Animal Care and Use Committee of Hiroshima University (approval number: A21-94). The experiments were also conducted in compliance with the National Institutes of Health Guidelines on the Use of Laboratory Animals. The study is reported in accordance with ARRIVE guidelines.

Experimental MGO-induced peritoneal fibrosis model

Peritoneal fibrosis was induced by intraperitoneal injection of 40 mM MGO (MP Biomedicals, Illkirch, France) in 2.5 mL saline for 3 weeks, with 5 days of administration per week and a 2-day break from administration as previously described ^63,64. A standard diet containing pemafibrate (K-877), provided by Kowa Corporation (Kowa Corporation, Tokyo, Japan), was started 3 weeks before beginning MGO administration to stabilize the blood concentration ⁶⁵. The mice were divided into three groups (n = 5 per group): intraperitoneal injection of 2.5 mL saline (control group), intraperitoneal injection of 40 mM MGO (MGO mice), and intraperitoneal injection of 40 mM MGO + oral administration of standard diet with 0.3 mg/kg/day pemafibrate (MGO + pemafibrate mice). All diaphragm samples were collected on day 21.

Peritoneal equilibrium test

The peritoneal equilibrium test (PET) was performed as previously described ⁶⁶. For the PET, 4 mL of PD solution (4.25% Dianeal; Baxter International, Deerfield, IL, USA) was injected into the abdominal cavity of mice under sedation with sodium pentobarbital anesthesia. Ten minutes later, ascitic fluid was collected. Blood samples were collected by cardiac puncture. Peritoneal permeability, determined by the glucose and blood urea nitrogen (BUN) concentrations, was calculated as the peritoneal absorption of glucose from the dialysate and the dialysate-to-plasma (D/P) ratio of BUN, respectively. Peritoneal samples were taken from the side opposite the injection.

Histology and immunohistochemistry

Histologic and immunohistochemical staining of 4-µm-thick tissue sections was performed as previously described ^64,67. The following primary antibodies were used: rabbit polyclonal anti-PPARα antibody (Abcam, Cambridge, UK, ab215270), mouse monoclonal anti-α-smooth muscle actin (anti-α-SMA) antibody (Sigma-Aldrich, St. Louis, MO, a2547), anti-fibroblast-specific protein 1 (anti-FSP-1) antibody (Abcam, ab41532), rabbit polyclonal anti-collagen I antibody (Abcam, ab34710), rabbit polyclonal anti-collagen III antibody (GeneTex, Irvine, CA, GTX102997), rabbit polyclonal anti-TGF-β1 antibody (Santa Cruz Biotechnology, Dallas, TX, sc-130348), mouse monoclonal anti-TNF-α antibody (Abcam, ab1793), mouse monoclonal anti-mouse CD68 antibody (Abcam, ab125212), anti-mouse CD163 antibody (Abcam, ab182422), and monoclonal anti-IFN-γ antibody (Abcam, ab267369). Based on the distinctive density and color of immunostaining in images, the number of stained cells and the area in the submesothelial zone above the abdominal muscle layer were quantified using ImageJ software (National Institutes of Health, Bethesda, MD) by examination of 10 randomly selected fields.

Cell culture

We isolated human peritoneal mesothelial cells (HPMCs) from human peritoneum as previously described ^66,68. The Medical Ethics Committee of Hiroshima Graduate School of Biomedical Science permitted harvesting of the omentum (E-2161). Written informed consent was acquired from each patient. We maintained the HPMCs in M199 medium (Life Technologies, Waltham, MA) including 10% fetal bovine serum and penicillin/streptomycin. HPMCs were seeded into six-well plates and grown to subconfluence. They were then treated with 80 ng/mL IFN-γ (Proteintech, Rosemont, IL) for 24 hours. Preincubation with pemafibrate (40 or 60 µmol/L) was conducted for 3 hours before the IFN-γ stimulation. In addition, the HPMCs were treated with 80 ng/mL IFN-γ (Proteintech) for 30 minutes to examine the expression of anti-phospho-cJun antibodies. Preincubation with pemafibrate (40 or 60 µmol/L) was conducted for 24 hours before the IFN-γ stimulation. We repeated the cell culture experiments five times.

THP-1 cells were obtained from the Cell Bank of the Chinese Academy of Sciences Type Culture Collection (Manassas, VA, USA). The cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum and penicillin/streptomycin (Biological Industries, Beit HaEmek, Israel). In all experiments, THP-1 monocytes were treated with 200 nM phorbol 12-myristate 13-acetate in six-well plates for 24 hours to transform them into adherent macrophages for culture. The THP-1 cells were then treated with 20 ng/mL IFN-γ (Proteintech) for 24 hours. Preincubation with pemafibrate (40 or 60 µmol/L) was conducted for 3 hours before the IFN-γ stimulation. We repeated the cell culture experiments five times.

Western blotting

Immunoblotting and detection of secreted fibronectin were performed as previously described ^69–71. The primary antibodies were CD163 (Abcam, ab182422), CD68 (Abcam, ab125212), anti-α-tubulin (Sigma-Aldrich, T9026), anti-TGF-β1 (Abcam, ab215715), anti-fibronectin (Sigma- Aldrich, F6140), anti-phospho-NF-κB (Cell Signaling Technology, 3033), anti-NF-κB (Cell Signaling Technology, 4764), anti-IκBα (Abcam, ab32518), anti-phospho-cJun (Cell Signaling Technology, 9164), anti-NLRP3 (Cell Signaling Technology, Danvers, MA, #15101), and anti-caspase1 (Abcam, ab207802). The intensity of each band was quantified using ImageJ software.

Enzyme-linked immunosorbent assay

TGF-β1 protein in mice peritoneal fluid samples, and TNF-α and IL-1β protein in THP-1 supernatant samples were measured with an enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN) in accordance with the manufacturer’s instructions. TNF-α and IL-1β concentrations were normalized to the total protein content of THP-1 cells.

Statistical analyses

All values are expressed as mean ± standard deviation. Between-group comparisons were made using Student’s t-test or one-way analysis of variance followed by Tukey’s post-hoc comparison as indicated in the figure legends. Values of P < 0.05 were considered statistically significant.

Acknowledgments

The authors thank Dr. Yoichi Sugiyama from JA Hiroshima General Hospital for his assistance with obtaining HPMCs and thank the Institute of Laboratory Animal Science at Hiroshima University for experimental mice. The authors also thank Angela Morben, DVM, ELS, from Edanz (https://jp.edanz.com/ac) for editing a draft of this manuscript.

Author Contributions

YS, RT, TM, and KS conceived the study and designed the experiments. YS, RT, TI, AT, YO, NI, and YM performed the experiments. YS, KS, AN, and TM analyzed the data. YS wrote the first draft of the manuscript, and KS, NI, AN, and TM edited the manuscript. All authors commented on previous versions of the manuscript and approved the final manuscript.

Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files.

Additional Information

Conflict of interest: The authors declare no conflicts of interest.

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Selective Activation of PPARα Mitigates Peritoneal Inflammation and Fibrosis through NLRP3 Inflammasome Suppression and Inflammation Modulation

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Abstract

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Introduction

Results

Enhancement of PPARα expression by pemafibrate suppressed MGO-induced peritoneal cell accumulation and thickening in mice

Pemafibrate inhibited MGO-induced fibrosis in mice with peritoneal fibrosis

Pemafibrate inhibited inflammatory cytokine production and macrophage accumulation in mice

Pemafibrate enhanced macrophage to an anti-inflammatory macrophage phenotype and inhibited inflammatory markers in THP-1 cells

Pemafibrate reduced peritoneal membrane functional impairments in mice with peritoneal fibrosis

Pemafibrate inhibited IFN-γ-induced fibrotic and inflammatory markers in HPMCs

Discussion

Methods

Animals

Experimental MGO-induced peritoneal fibrosis model

Peritoneal equilibrium test

Histology and immunohistochemistry

Cell culture

Western blotting

Enzyme-linked immunosorbent assay

Statistical analyses

Declarations

References

Additional Declarations

Supplementary Files

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